Tag Archives: genome

This research isn’t quite as exciting as the title promises but it is important as it attempts to answer some fundamental questions about Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas).proteins. From a June 13, 2018 news item on phys.org,

Recently published research from the University of Georgia and UConn Health [University of Connecticu Health Center] provides new insight about the basic biological mechanisms of the RNA-based viral immune system known as CRISPR-Cas.

CRISPR-Cas, short for Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated, is a defense mechanism that has evolved in bacteria and archaea that these single celled organisms use to ward off attacks from viruses and other invaders. When a bacterium is attacked by a virus, it makes a record of the virus’s DNA by chopping it up into pieces and incorporating a small segment of the invader’s DNA into its own genome. It then uses this DNA to make RNAs that bind with a bacterial protein that then kills the viral DNA.

The system has been studied worldwide in hopes that it can be used to edit genes that predispose humans to countless diseases, such as diabetes and cancer. However, to reach this end goal, scientists must gain further understanding of the basic biological process that leads to successful immunity against the invading virus.

Distinguished Research Professor of Biochemistry and Molecular Biology in UGA’s Franklin College of Arts and Sciences and principal investigator for the project Michael Terns and UGA postdoctoral fellow Masami Shiimori collaborated with Brenton Graveley and Sandra Garrett at UConn Health to sequence millions of genomes to learn more about the process. Graveley is professor and chair of the Department of Genetics and Genome Sciences and associate director of the Institute for Systems Genomics at UConn Health, and Garrett is a postdoctoral fellow in his laboratory.

“This research is more fundamental and basic than studies that are trying to determine how to use CRISPR for therapeutic or biomedical application,” said Terns. “Our study is about the unique first step in the process, known as adaptation, where fragments of DNA are recognized and integrated into the host genome and provide immunity for future generations.”

Previously, researchers did not understand how the cell recognized the virus as an invader, nor which bacterial proteins were necessary for successful integration and immunity.

“In this project we were able to determine how the bacterial immune system creates a molecular memory to remove harmful viral DNA sequences and how this is passed down to the bacterial progeny,” said Graveley.

By looking at patterns in the data, the researchers discovered several new findings about how two previously poorly characterized Cas4 proteins function in tandem with Cas1 and Cas2 proteins found in all CRISPR-Cas systems.

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In this initial adaptation phase, one of two different Cas4 proteins recognizes a signaling placeholder in the sequence that occurs adjacent to the snippet of DNA that is excised.

When the Cas1 and Cas2 proteins are present in the cell with either of two Cas4 protein nucleases, Cas4-1 and Cas4-2, they act like the generals of this army-based immune system, communicating uniform sized clipped DNA fragments, directions on where to go next and ultimately instructions that destroy the lethal DNA fragment.

In order for a cell to successfully recognize and excise strands of DNA, incorporate them into its own genome and achieve immunity, the Cas4 proteins must be present in conjunction with the Cas1 and Cas2 proteins.

“Cas4 is present in many CRISPR-Cas systems, but the roles of the proteins were mysterious,” said Terns. “In our system, there are two Cas4 proteins that are essential for governing this process so that functional RNAs are made and immunity is conferred”

To achieve these findings, the research team from the University of Georgia created strains of archaeal organisms with key genetic deletions.

Hundreds of millions of DNA fragments captured in the CRISPR loci were sent to the Graveley lab in Farmington, Connecticut, where they were sequenced with the Illumina MiSeq system. The researchers then used supercomputing for bioinformatics analysis and data interpretation.

While there is still much to learn about the biological mechanisms involved in CRISPR-Cas systems, this research tells scientists more about the way these proteins work together to save the cell and achieve immunity.

“The data are so clear. We sequenced millions and millions of DNA fragments captured in CRISPR loci in different genetic strains and found the same results consistently,” he said.

A November 21, 2017 news item on Nanowerk describes a rather extraordinary (to me, anyway) approach to using CRRISP ( Clustered Regularly Interspaced Short Palindromic Repeats)-CAS9 (Note: A link has been removed),

A team of scientists led by Virginia Commonwealth University physicist Jason Reed, Ph.D., have developed new nanomapping technology that could transform the way disease-causing genetic mutations are diagnosed and discovered. Described in a study published today [November 21, 2017] in the journal Nature Communications (“DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle”), this novel approach uses high-speed atomic force microscopy (AFM) combined with a CRISPR-based chemical barcoding technique to map DNA nearly as accurately as DNA sequencing while processing large sections of the genome at a much faster rate. What’s more–the technology can be powered by parts found in your run-of-the-mill DVD player.

The human genome is made up of billions of DNA base pairs. Unraveled, it stretches to a length of nearly six feet long. When cells divide, they must make a copy of their DNA for the new cell. However, sometimes various sections of the DNA are copied incorrectly or pasted together at the wrong location, leading to genetic mutations that cause diseases such as cancer. DNA sequencing is so precise that it can analyze individual base pairs of DNA. But in order to analyze large sections of the genome to find genetic mutations, technicians must determine millions of tiny sequences and then piece them together with computer software. In contrast, biomedical imaging techniques such as fluorescence in situ hybridization, known as FISH, can only analyze DNA at a resolution of several hundred thousand base pairs.

Reed’s new high-speed AFM method can map DNA to a resolution of tens of base pairs while creating images up to a million base pairs in size. And it does it using a fraction of the amount of specimen required for DNA sequencing.

“DNA sequencing is a powerful tool, but it is still quite expensive and has several technological and functional limitations that make it difficult to map large areas of the genome efficiently and accurately,” said Reed, principal investigator on the study. Reed is a member of the Cancer Molecular Genetics research program at VCU Massey Cancer Center and an associate professor in the Department of Physics in the College of Humanities and Sciences.

“Our approach bridges the gap between DNA sequencing and other physical mapping techniques that lack resolution,” he said. “It can be used as a stand-alone method or it can complement DNA sequencing by reducing complexity and error when piecing together the small bits of genome analyzed during the sequencing process.”

IBM scientists made headlines in 1989 when they developed AFM technology and used a related technique to rearrange molecules at the atomic level to spell out “IBM.” AFM achieves this level of detail by using a microscopic stylus — similar to a needle on a record player — that barely makes contact with the surface of the material being studied. The interaction between the stylus and the molecules creates the image. However, traditional AFM is too slow for medical applications and so it is primarily used by engineers in materials science.

“Our device works in the same fashion as AFM but we move the sample past the stylus at a much greater velocity and use optical instruments to detect the interaction between the stylus and the molecules. We can achieve the same level of detail as traditional AFM but can process material more than a thousand times faster,” said Reed, whose team proved the technology can be mainstreamed by using optical equipment found in DVD players. “High-speed AFM is ideally suited for some medical applications as it can process materials quickly and provide hundreds of times more resolution than comparable imaging methods.”

Increasing the speed of AFM was just one hurdle Reed and his colleagues had to overcome. In order to actually identify genetic mutations in DNA, they had to develop a way to place markers or labels on the surface of the DNA molecules so they could recognize patterns and irregularities. An ingenious chemical barcoding solution was developed using a form of CRISPR technology.

CRISPR has made a lot of headlines recently in regard to gene editing. CRISPR is an enzyme that scientists have been able to “program” using targeting RNA in order to cut DNA at precise locations that the cell then repairs on its own. Reed’s team altered the chemical reaction conditions of the CRISPR enzyme so that it only sticks to the DNA and does not actually cut it.

“Because the CRISPR enzyme is a protein that’s physically bigger than the DNA molecule, it’s perfect for this barcoding application,” Reed said. “We were amazed to discover this method is nearly 90 percent efficient at bonding to the DNA molecules. And because it’s easy to see the CRISPR proteins, you can spot genetic mutations among the patterns in DNA.”

To demonstrate the technique’s effectiveness, the researchers mapped genetic translocations present in lymph node biopsies of lymphoma patients. Translocations occur when one section of the DNA gets copied and pasted to the wrong place in the genome. They are especially prevalent in blood cancers such as lymphoma but occur in other cancers as well.

While there are many potential uses for this technology, Reed and his team are focusing on medical applications. They are currently developing software based on existing algorithms that can analyze patterns in sections of DNA up to and over a million base pairs in size. Once completed, it would not be hard to imagine this shoebox-sized instrument in pathology labs assisting in the diagnosis and treatment of diseases linked to genetic mutations.

In a May 5, 2013 posting I featured a Kickstarter campaign for a synthetic biology project focused on plants that emit light in the dark. I also mentioned Eduardo Kac (pronounced Katz) and his art project/transgenic bunny called Alba. At the time, I did not realize that Alba had been declared dead in 2002 adding more controversy to an already controversial topice according to Kristen Philipkoski in an Aug. 12, 2002 article (how did I miss this article in 2013?) for Wired magazine (Note: Links have been removed),

Alba, the glowing rabbit that made headlines two years ago for being, well, a glowing rabbit, has met an untimely death, according to the French researcher who genetically engineered her.

Alba the glowing rabbit was 4 years old. Or 2-1/2, depending on who’s talking.

The bunny died about a month ago for reasons that are not clear, said Louis-Marie Houdebine, a genetic researcher at France’s National Institute of Agronomic Research.

“I was informed one day that bunny was dead without any reason,” Houdebine said. “So, rabbits die often. It was about 4 years old, which is a normal lifespan in our facilities.”

Alba was an albino rabbit engineered by splicing the green fluorescent protein (GFP) of a jellyfish into her genome. Houdebine said he did not believe the GFP gene played a role in the animal’s demise.

Eduardo Kac, the artist who created a flurry by making her a work of art, doesn’t buy it, however.

First, Alba’s not 4, she’s 2-1/2, Kac says (a rabbit’s lifespan is up to 12 years), because she was bred by Houdebine specifically for him in January 2000.

Houdebine says he simply picked a rabbit with a gentle disposition that was already in his lab.

Second, he believes Houdebine might be declaring the bunny gone in order to put an end to a two-year, unwelcome barrage of media attention.

If she really is dead, Kac will never realize the final phase of his project, which was to take Alba home and keep her as a pet.

Kac says he and Houdebine originally collaborated on the GFP bunny project, until Houdebine’s director put the kibosh on it.

“My director did not understand,” Houdebine said. “He said I should not give the rabbit (to someone) outside the lab.”

Houdebine said that yes, they spoke about preliminary plans for Kac to use the bunny for his project and take it to an art show in Avignon. But he denies he bred an animal specifically for Kac.

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Houdebine says he would not have agreed to engineer one animal specifically for any artist.

This disputed point has led fellow artists and critics to question whether Kac can rightly take credit for the Alba project.

But Kac insists that Houdebine did, in fact, agree to make the bunny specifically for him.

Kac found out sometime in mid-2000 that Houdebine’s director had a problem with the project and would not allow the rabbit to be taken from the lab.

Houdebine was initially apologetic, Kac said. But after an article ran on the front page of the Boston Globe on Sept. 17, 2000, their relationship cooled.

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Houdebine and his director were opposed to the now-famous, brilliantly glowing photograph of Alba. They and other researchers say the rabbit doesn’t actually glow so brightly and uniformly.

“Kac fabricated data for his personal use,” Houdebine said. “This is why we totally stopped any contact with him.”

“The scientific fact is that the rabbit is not green,” he said. “He should have never published that. This was very disagreeable for me.”

Kac believes the scientists were simply afraid of public criticism. Meanwhile, he wanted to do the opposite – to encourage discourse on the transgenic rabbit.

“This director refuses to participate openly in a debate about what is done with public money,” he said. “It’s very easy to fear and reject what you don’t know. As long as they continue to isolate themselves, this mistrust will continue.”

The eyes and ears of the rabbit are green under ultraviolet light, Houdebine said, but the fur does not glow, because it’s dead tissue that doesn’t express the gene. Only if the rabbit were shaved would the body glow, he said.

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Philipkosk’s article provides some insight into the interface between art and science and is worth reading in its entirety if you have the time.

I’ve also found an update for the glowing plants Kickstarter campaign in an April 20, 2017 article by Sarah Zhang for The Atlantic (Note: Links have been removed),

The latest update came quietly on Tuesday night [April 18, 2017?]. “We’re sorry to say that we have reached a significant transition point,” wrote the Glowing Plant project’s creator, Antony Evans. This “transition point” was more of an endpoint: The project had run out of money. The quest to genetically engineer a glow-in-the-dark plant was no more.

Four years ago, the Glowing Plant project raised nearly half a million dollars on Kickstarter, easily blowing past its initial ask of $65,000. Of course it did. The vision it presented was such potent fantasy. “What if,” Evans asked over swelling music in the pitch video, “we use trees to light our streets instead of street lamps?” What if you could get lighting without electricity? What if the natural world glowed like in Avatar?

This romantic vision so perfectly encapsulated the promises of synthetic biology, a field that treats the natural world as another system to be designed and engineered. In this case, synthetic biology became a possible solution to one of the world’s most pressing energy problems: electricity generation. Plus, it sounded really damn cool.

The Kickstarter campaign only promised a small, potted glowing plant to it backers, and I doubt many backers actually harbored illusions about trees lighting up the night sky soon. But backing the project was a small way to buy into a much grander vision.

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At a time when “genetically modified organism,” or GMO, is such a poisoned phrase, the project’s crowdfunding success seemed to suggest that a pervasive if vague distrust of genetic modification might be countered by the sense of wonder for a glowing plant. (As the Kickstarter campaign grew, though, environmental groups raised questions and the crowdfunding site later banned giving away genetically modified organisms.)

The team also encountered the hard realities of engineering even a small plant that glows. “We did not anticipate some of the unknown technical challenges that we would get into,” Evans told me. (Plenty of scientists at the time were skeptical of the project’s timeline, though.) Evans is an MBA with a background in mobile apps, though his two original cofounders, who have both since left the project, had backgrounds in synthetic biology.

To get the plant to glow well, the research team had to insert six genes. But they never could get all six in at once. At best, some plants glowed very dimly. (The photo above of the glowing plant is a long exposure, making it appear much brighter than it actually is.) Evans says that he realizes now trying to insert six genes into a complex organism like a plant—rather than single-celled bacteria or yeast—was premature.

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“I’m really afraid of disappointing that 16-year-old who saw this and imagined a bright wonderful future, of jading and disappointing people,” he says. Despite a few angry backers asking for a refund, most of the comments under the Kickstarter update so far have been supportive. The project had been providing regular, detailed updates on the difficulty of engineering the plants. The latest update was its 67th.

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Zhang’s article goes on to detail other synthetic biology projects, which are showing some promise.

When you take this work into consideration with CRISPR-CAS9 and the beginnings of genetic germline editing, the question has to be asked: Will public discussion (if there’s any) be considered upstream (early in the process) or downstream (after the work has been done)? Public engagement professionals tend to favour upstream discussions, i.e., before people start demanding fear-based policy.

Apparently, not all premium chocolate is actually premium, like wine, expensive, premium product can be mixed with a more common variety to be sold at the higher, premium price. Now, scientists in a collaboration which spans the US, China, and Trinidad and Tobago have found a way to authenticate premium chocolate according to a Jan. 15, 2014 news release on EurekAlert,

For some people, nothing can top a morsel of luxuriously rich, premium chocolate. But until now, other than depending on their taste buds, chocolate connoisseurs had no way of knowing whether they were getting what they paid for. In ACS’ Journal of Agricultural and Food Chemistry, scientists are reporting, for the first time, a method to authenticate the varietal purity and origin of cacao beans, the source of chocolate’s main ingredient, cocoa.

Dapeng Zhang and colleagues note that lower-quality cacao beans often get mixed in with premium varieties on their way to becoming chocolate bars, truffles, sauces and liqueurs. But the stakes for policing the chocolate industry are high. It’s a multi-billion dollar global enterprise, and in some places, it’s as much art as business. There’s also a conservation angle to knowing whether products are truly what confectioners claim them to be. The ability to authenticate premium and rare varieties would encourage growers to maintain cacao biodiversity rather than depend on the most abundant and easiest to grow trees. Researchers have found ways to verify through genetic testing the authenticity of many other crops, including cereals, fruits, olives, tea and coffee, but those methods aren’t suitable for cacao beans. Zhang’s team wanted to address this challenge.

Applying the most recent developments in cacao genomics, they were able to identify a small set of DNA markers called SNPs (pronounced “snips”) that make up unique fingerprints of different cacao species. The technique works on single cacao beans and can be scaled up to handle large samples quickly. “To our knowledge, this is the first authentication study in cacao using molecular markers,” the researchers state.

Here’s an image, provided by the researchers, illustrating their work,

This story reminded me that coffee too is sold at premium prices. Billed as the most expensive coffee in the world, Kopi Luwak, is harvested, so they say, from civet excrement and I have to wonder how anyone could authenticate that a bean had actually passed through a civet’s gastrointestinal tract and out the other end. I’ve also wondered how the practice of plucking coffee beans from civet excrement started (from the Kopi Luwak Wikipedia essay; Note: Links have been removed) here’s an answer to the second question,

The origin of kopi luwak is closely connected with the history of coffee production in Indonesia. In the early 18th century the Dutch established the cash-crop coffee plantations in their colony in the Dutch East Indies islands of Java and Sumatra, including Arabica coffee introduced from Yemen. During the era of Cultuurstelsel (1830—1870), the Dutch prohibited the native farmers and plantation workers from picking coffee fruits for their own use. Still, the native farmers wanted to have a taste of the famed coffee beverage. Soon, the natives learned that certain species of musang or luwak (Asian Palm Civet) consumed the coffee fruits, yet they left the coffee seeds undigested in their droppings. The natives collected these luwaks’ coffee seed droppings, then cleaned, roasted and ground them to make their own coffee beverage.[11] The fame of aromatic civet coffee spread from locals to Dutch plantation owners and soon became their favourite, yet because of its rarity and unusual process, the civet coffee was expensive even during the colonial era.[citation needed]

I guess that in the future when you eat premium chocolate you can be sure that you’ve gotten what you paid for. As for coffee, I’m sure that industry is working on its authentication processes too and in the meantime, you’ll have to rely on your palate.

Researchers in Canada and the US have resolved a question about DNA and structural protein. From the Oct. 4, 2012 news release on EurekAlert,

Scientists in Canada and the United States have used three-dimensional imaging techniques to settle a long-standing debate about how DNA and structural proteins are packaged into chromatin fibres. The researchers, whose findings are published in EMBO [European Molecular Biology Organization] reports, reveal that the mouse genome consists of 10-nm chromatin fibres but did not find evidence for the wider 30-nm fibres that were previously thought to be important components of the DNA architecture.

Scientists were trying to understand how DNA can be packed into a cell,

“DNA is an exceptionally long molecule that can reach several metres in length. This means it needs to be packaged into a highly compact state to fit within the limited space of the cell nucleus,” said David Bazett-Jones, Senior Scientist at the Hospital for Sick Children, Toronto, and Professor at the University of Toronto, Canada. “For the past few decades, scientists have favoured structural models for chromatin organization where DNA is first wrapped around proteins in nucleosomes. In one possible model, the strand of repeating nucleosomes is wrapped further into a higher-order thick 30-nm fibre. In a second model, the 30-nm fibre is not required to compact the DNA. Differences between these models have implications for the way the cell regulates the transcription of genes.”

Scientists offer reasons for why they concluded Previous studies have suggested for a 30-nm fibre model in earlier studies,

The researchers offer several reasons for the observation of wider fibres in earlier studies. In some cases, the conditions outside of the cell, including those used in earlier studies where chromatin was extracted from the cell, may have given rise to structural artifacts. For some of the earlier spectroscopic studies, it may even be a question of poor resolution of existing 10-nm fibres.

Here’s what the scientists found,

“Our results revealed that the 30-nm chromatin fibre model is not consistent with the structure we found in our three-dimensional spectroscopic images,” said Bazett-Jones. “It was previously thought that the transition between thinner and thicker fibres represented a change from an active to repressed state of chromatin. However, our inability to detect 30-nm fibres in the mouse genome leads us to conclude that the transcriptional machinery has widespread access to the DNA packaged into chromatin fibres.”

The results are consistent with recent studies of the human genome which suggest that approximately 80% of the genome contains elements that are linked to biological function. Access to enhancers, promoters and other regulatory sequences on such a wide region of the genome means that all of these sites must be accessible. The 10-nm model of chromatin fibres provides sufficient access to DNA to allow potential target sites to be reached. The 30-nm model would not accommodate such widespread access.